PROJECT SUMMARY This project ?Diagnostics via Rapid Enrichment, Identification, and Phenotypic Antibiotic Susceptibility Testing of Pathogens from Blood,? submitted under RFA-AI-17-014 ?Partnerships for Development of Clinically Useful Diagnostics for Antimicrobial-Resistant Bacteria (R01)? will develop a rapid, sensitive, and specific diagnostic platform for the culture-independent identification and determination of antimicrobial susceptibility and/or resistance of bacterial pathogens. The indiscriminate use and misuse of antibiotics has led to an impending global health crisis: the development of widespread antibiotic resistance. Bloodstream infections (BSI) are particularly significant with respect to their clinical impact: Severe sepsis strikes more than one million Americans every year, and 15 to 30 percent of those people die. In the absence of a rapid and reliable antibiotic susceptibility test (AST), health care providers often resort to broad-spectrum antibiotics, further escalating the development of drug-resistant strains. Halting the emergence and spread of antibiotic resistant organisms and providing appropriate life-saving therapy requires point-of-care (POC) diagnostics that can rapidly measure the drug susceptibility of a pathogen directly from clinical samples, eliminating the lengthy current procedures that require microbial growth. This project addresses a critical unmet need for rapid diagnostics that can both identify and determine the antimicrobial resistance profile of microbial pathogens. To address this problem, we propose to develop a combination of highly innovative technologies to (1) concentrate viable, low-abundance pathogens directly from blood without a culture step; (2) demonstrate the compatibility of the enrichment technology with the rapid and specific multiplexed detection of pathogens; (3) validate the compatibility of enrichment with an innovative, digital method of determining phenotypic antibiotic susceptibility; and (4) demonstrate that the entire workflow of pathogen concentration, identification, and phenotypic antibiotic susceptibility testing can be achieved in under 2 hours directly from human blood. This multidisciplinary project will be carried out by a highly experienced industrial research team with outstanding academic and clinical collaborators, and will create a new paradigm in diagnosis and treatment of bloodstream infections directly from clinical samples at the point- of-care. Countless lives will be saved and the spread of antibiotic resistance will be halted.